Inhaled mRNA nanoparticles dual-targeting cancer cells and macrophages in the lung for effective transfection.

Messenger RNA (mRNA)-based therapeutics are transforming the landscapes of medicine, yet targeted delivery of mRNA to specific cell types while minimizing off-target accumulation remains challenging for mRNA-mediated therapy. In this study, we report an innovative design of a cationic lipid- and hyaluronic acid-based, dual-targeted mRNA nanoformulation that can display the desirable stability and efficiently transfect the targeted proteins into lung tissues. More importantly, the optimized dual-targeted mRNA nanoparticles (NPs) can not only accumulate primarily in lung tumor cells and inflammatory macrophages after inhalation delivery but also efficiently express any desirable proteins (e.g., p53 tumor suppressor for therapy, as well as luciferase and green fluorescence protein for imaging as examples in this study) and achieve efficacious lung tissue transfection in vivo. Overall, our findings provide proof-of-principle evidence for the design and use of dual-targeted mRNA NPs in homing to specific cell types to up-regulate target proteins in lung tissues, which may hold great potential for the future development of mRNA-based inhaled medicines or vaccines in treating various lung-related diseases.

Monovalent SARS-COV-2 mRNA vaccine using optimal UTRs and LNPs is highly immunogenic and broadly protective against Omicron variants.

The emergence of highly transmissible severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOCs) that are resistant to the current COVID-19 vaccines highlights the need for continued development of broadly protective vaccines for the future. Here, we developed two messenger RNA (mRNA)-lipid nanoparticle (LNP) vaccines, TU88mCSA and ALCmCSA, using the ancestral SARS-CoV-2 spike sequence, optimized 5' and 3' untranslated regions (UTRs), and LNP combinations. Our data showed that these nanocomplexes effectively activate CD4+ and CD8+ T cell responses and humoral immune response and provide complete protection against WA1/2020, Omicron BA.1 and BQ.1 infection in hamsters. Critically, in Omicron BQ.1 challenge hamster models, TU88mCSA and ALCmCSA not only induced robust control of virus load in the lungs but also enhanced protective efficacy in the upper respiratory airways. Antigen-specific immune analysis in mice revealed that the observed cross-protection is associated with superior UTRs [Carboxylesterase 1d (Ces1d)/adaptor protein-3ß (AP3B1)] and LNP formulations that elicit robust lung tissue-resident memory T cells. Strong protective effects of TU88mCSA or ALCmCSA against both WA1/2020 and VOCs suggest that this mRNA-LNP combination can be a broadly protective vaccine platform in which mRNA cargo uses the ancestral antigen sequence regardless of the antigenic drift. This approach could be rapidly adapted for clinical use and timely deployment of vaccines against emerging and reemerging VOCs.

CRISPR-Cas9 Genome Editing Allows Generation of the Mouse Lung in a Rat.

Rationale: Recent efforts in bioengineering and embryonic stem cell (ESC) technology allowed the generation of ESC-derived mouse lung tissues in transgenic mice that were missing critical morphogenetic genes. Epithelial cell lineages were efficiently generated from ESC, but other cell types were mosaic. A complete contribution of donor ESCs to lung tissue has never been achieved. The mouse lung has never been generated in a rat. Objective: We sought to generate the mouse lung in a rat. Methods: Clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 genome editing was used to disrupt the Nkx2-1 gene in rat one-cell zygotes. Interspecies mouse-rat chimeras were produced by injection of wild-type mouse ESCs into Nkx2-1-deficient rat embryos with lung agenesis. The contribution of mouse ESCs to the lung tissue was examined by immunostaining, flow cytometry, and single-cell RNA sequencing. Measurements and Main Results: Peripheral pulmonary and thyroid tissues were absent in rat embryos after CRISPR-Cas9-mediated disruption of the Nkx2-1 gene. Complementation of rat Nkx2-1-/- blastocysts with mouse ESCs restored pulmonary and thyroid structures in mouse-rat chimeras, leading to a near-99% contribution of ESCs to all respiratory cell lineages. Epithelial, endothelial, hematopoietic, and stromal cells in ESC-derived lungs were highly differentiated and exhibited lineage-specific gene signatures similar to those of respiratory cells from the normal mouse lung. Analysis of receptor-ligand interactions revealed normal signaling networks between mouse ESC-derived respiratory cells differentiated in a rat. Conclusions: A combination of CRISPR-Cas9 genome editing and blastocyst complementation was used to produce mouse lungs in rats, making an important step toward future generations of human lungs using large animals as "bioreactors."

Severe Acute Respiratory Syndrome Coronavirus 2 Infection Alters Mediators of Lung Tissue Remodeling In Vitro and In Vivo.

BACKGROUND: Altered mediators of airway tissue remodeling such as matrix metalloproteinases (MMPs) in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection may contribute to morbidity in coronavirus disease 2019 (COVID-19); however, the differential impact of SARS-CoV-2 variants of concern (VOCs) on MMPs is unknown. METHODS: Using both in vitro human airway cell culture model and in vivo transgenic mouse model of SARS-CoV-2 infection, we studied the differential effect of SARS-CoV-2 VOCs on expression of key MMPs and inflammatory mediators in airway cells and tissues. RESULTS: The most consistent findings with all SARS-CoV-2 variants in infected compared to uninfected human bronchial epithelial cell air-liquid interface cultures were the SARS-CoV-2-induced increases in MMP-12 and tissue inhibitor of MMPs. Infection with both SARS-CoV-2 wild type and SARS-CoV-2 Delta variant over 3 days postinfection (dpi) and with Beta variant over 7 dpi increased lung tissue levels of MMP-9 compared to uninfected mice. Overall, SARS-CoV-2 variants had differential dose-dependent impact on secretion of MMP-1, MMP-2, MMP-9, and MMP-12 that varied at the protein versus the gene level and in the early noninflammatory compared to late inflammatory phase of infection. CONCLUSIONS: We provide novel mechanistic insight that the differential impact of SARS-CoV-2 variants on severity of COVID-19 may partially be attributed to unique changes in MMPs.

Single-Cell Profiling of the Differential In Vivo Impact of Severe Acute Respiratory Syndrome Coronavirus 2 Infection Among Lung Tissue Cell Subtypes at the Protein Level.

The complexity of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and its variants in lung cells can truly be characterized only at the tissue and protein levels among unique cell subtypes. However, in vivo data are limited due to lack of accessible human tissues. Using a transgenic mouse model of SARS-CoV-2 infection and flow cytometry, we provide in vivo novel insight at the protein level that the differential impact of SARS-CoV-2 (Wuhan strain) and its B.1.617.2 (Delta) and BA.1 (Omicron) variants on lung may be attributed to differential patterns of viral protein levels among ciliated airway cells, alveolar types 1 and 2 cells, immune cells, and endothelial lung cells.

A Rapid Human Lung Tissue Dissociation Protocol Maximizing Cell Yield and Minimizing Cellular Stress.

The human lung is a complex organ comprised of diverse populations of epithelial, mesenchymal, vascular and immune cells, which gains even greater complexity during disease states. To effectively study the lung at a single cell level, a dissociation protocol that achieves the highest yield of viable cells of interest with minimal dissociation-associated protein or transcription changes key. Here, we detail a rapid collagenase-based dissociation protocol (Col-Short), which provides a high-yield single cell suspension suitable for a variety of downstream applications. Diseased human lung explants were obtained and dissociated through the Col-Short protocol and compared to four other dissociation protocols. Resulting single cell suspensions were then assessed with flow cytometry, differential staining, and quantitative real-time PCR to identify major hematopoietic and non-hematopoietic cell populations, as well as their activation states. We observed that the Col-Short protocol provides the greatest number of cells per gram of lung tissue with no reduction in viability when compared to previously described dissociation protocols. Col-Short had no observable surface protein marker cleavage as well as lower expression of protein activation markers and stress-related transcripts compared to four other protocols. The Col-Short dissociation protocol can be used as a rapid strategy to generate single cells for respiratory cell biology research.

Bafi A1 inhibits nano-copper oxide-induced mitochondrial damage by reducing the release of copper from lysosomes.

This study demonstrated that both copper oxide nanoparticles (CuO-NPs) and copper nanoparticles (Cu-NPs) can cause swelling, inflammation, and cause damage to the mitochondria of alveolar type II epithelial cells in mice. Cellular examinations indicated that both CuO-NPs and Cu-NPs can reduce cell viability and harm the mitochondria of human bronchial epithelial cells, particularly Beas-2B cells. However, it is clear that CuO-NPs exhibit a more pronounced detrimental effect compared with Cu-NPs. Using bafilomycin A1 (Bafi A1), an inhibitor of lysosomal acidification, was found to enhance cell viability and alleviate mitochondrial damage caused by CuO-NPs. Additionally, Bafi A1 also reduces the accumulation of dihydrolipoamide S-acetyltransferase (DLAT), a marker for mitochondrial protein toxicity, induced by CuO-NPs. This observation suggests that the toxicity of CuO-NPs depends on the distribution of copper particles within cells, a process facilitated by the acidic environment of lysosomes. The release of copper ions is thought to be triggered by the acidic conditions within lysosomes, which aligns with the lysosomal Trojan horse mechanism. However, this association does not seem to be evident with Cu-NPs.

Chronic inhalation of H2S in low concentration induces immunotoxicity and inflammatory effects in lung tissue of rats.

Hydrogen sulfide (H2S) is a typical odour compound mainly causing respiratory and central nervous system symptoms. However, the immunotoxicity of inhaled H2S and the underlying mechanisms remain largely unknown. In this study, a low-dose inhalation exposure to H2S was arranged to observe inflammatory response and immunotoxicity in lung tissue of rats. Low concentrations of H2S exposure affected the immune level of pulmonary tissue and peripheral blood. Significant pathological changes in lung tissue in the exposure group were observed. At low concentration, H2S not only induced the upregulation of AQP-4 and MMP-9 expression but also stimulated immune responses, initiating various anti-inflammatory and inflammatory factors, altering tissue homeostatic environments. The TNF and chemokine signaling pathway played an important role which can promote the deterioration of pulmonary inflammatory processes and lead to lung injury and fibrosis. Excessive immune response causes an inflammatory effect and blood-gas barrier damage. These data will be of value in evaluating future occupational health risks and providing technical support for the further development of reliable, sensitive, and easy-to-use screening indicators of exposure injury.

Olea europaea L. leaf extract mitigates pulmonary inflammation and tissue destruction in Wistar rats induced by concurrent exposure to noise and toluene.

This study aimed to investigate the effects of combined exposure to noise (85 dB(A)) and inhaled Toluene (300 ± 10 ppm) on rat lung health. It also aimed to assess the potential therapeutic effects of Olea europaea L. leaves extract (OLE) (40 mg/kg/day) using biochemical, histopathological, and immunohistochemical (IHC) analyses, as well as determination of pro-inflammatory cytokines (TNF-α and IL-1ß), and in silico Docking studies. The experiment involved forty-two male Wistar rats divided into seven groups, each exposed to a 6-week/6-hour/day regimen of noise and Toluene. The groups included a control group, rats co-exposed to noise and Toluene, and rats co-exposed to noise and Toluene treated with OLE for different durations. The results indicated that noise and Toluene exposure led to structural damage in lung tissue, oxidative harm, and increased levels of pro-inflammatory cytokines (TNF-α and IL-1ß). However, the administration of OLE extract demonstrated positive effects in mitigating these adverse outcomes. OLE treatment reduced lipid peroxidation and enhanced the activities of catalase and superoxide dismutase, indicating its anti-oxidant properties. Furthermore, OLE significantly decreased the levels of pro-inflammatory cytokines compared to the groups exposed to noise and Toluene without OLE treatment. Moreover, the in silico investigation substantiated a robust affinity between COX-2 and OLE components, affirming the anti-inflammatory activity. Overall, our findings suggest that OLE possesses anti-inflammatory and anti-oxidative properties that mitigate the adverse effects of concurrent exposure to noise and Toluene.

Effects of Bosentan on Hypoxia, Inflammation and Oxidative Stress in Experimental Blunt Thoracic Trauma Model.

Background and Objectives: In this study, we aimed to investigate the effects of bosentan, an endothelin receptor antagonist, on endothelin-1 (ET-1), hypoxia-inducible factor-1 (HIF-1), nuclear factor-kappa B (NF-κB), and tumor necrosis factor (TNF)-α as inflammation markers, pro-oxidant antioxidant balance (PAB), and total antioxidant capacity (TAC) levels as oxidative stress parameters in lung tissues of rats in an experimental model of pulmonary contusion (PC) induced by blunt thoracic trauma. Materials and Methods: Thirty-seven male Sprague-Dawley rats were divided into five groups. C: The control group (n = 6) consisted of unprocessed and untreated rats. PC3 (n = 8) underwent 3 days of PC. PC-B3 (n = 8) received 100 mg/kg bosentan and was given orally once a day for 3 days. The PC7 group (n = 7) underwent 7 days of PC, and PC-B7 (n = 8) received 100 mg/kg bosentan and was given orally once a day for 7 days. Results: ET-1, NF-κB, TNF-α, HIF-1α, and PAB levels were higher, while TAC activity was lower in all groups compared with the control (p < 0.05). There was no significant difference in ET-1 and TNF-α levels between the PC-B3 and PC-B7 groups and the control group (p < 0.05), while NF-κB, HIF-1α, and PAB levels were still higher in both the PC-B3 and PC-B7 groups than in the control group. Bosentan decreased ET-1, NF-κB, TNF-α, HIF-1α, and PAB and increased TAC levels in comparison to the nontreated groups (p < 0.05). Conclusions: Bosentan decreased the severity of oxidative stress in the lungs and reduced the inflammatory reaction in rats with PC induced by blunt thoracic trauma. This suggests that bosentan may have protective effects on lung injury mechanisms by reducing hypoxia, inflammation, and oxidative stress. If supported by similar studies, bosentan can be used in both pulmonary and emergency clinics to reduce ischemic complications, inflammation, and oxidative stress in some diseases that may be accompanied by ischemia.

[Study on multiple organ injury induced by Haematitum in mice].

Haematitum is a commonly used mineral medicine. It is toxic, as recorded in the second volume of Chinese Materia Medica. Therefore, it should not be taken for a long time. In this study, the effects of Haematitum and calcined Haematitum on multiple organ injuries in mice were investigated, and the mechanism of the toxicity of the related organs was explored by metabolomics. The mice were randomly divided into the control group, Haematitum low-dose group(ZS-L group), Haematitum high-dose group(ZS-H group), and calcined Haematitum high-dose group(DZS-H group), with 12 mice in each group. Haematitum decoction was given by continuous intragastric administration for 10 days. Then the life situation was observed, and samples were taken to detect various indicators. The results showed that the ZS-H group showed obvious toxicity, with different degrees of toxicity damage in the intestinal tract,liver, spleen, and lung. ZS-L group had no toxic reaction. The toxicity of the DZS-H group was significantly reduced, and only the lung was damaged. Metabolomics technology was used to detect the lung tissue of mice in the control group and the ZS-H group, and a total of 15 kinds of significant difference metabolites were detected, mainly involved in choline metabolism in cancer, sphingolipid metabolism, and glycerophospholipid metabolism. Immunohistochemical results showed that the INSIG1 protein expression level in the lung tissue of mice in the ZS-H group was significantly higher than that in the control group. In summary, large doses and long-time use of Haematitum decoction will cause a variety of organ damage, and the same dose of calcined Haematitum is less toxic than Haematitum. In addition, a low dose of Haematitum has no obvious toxic effect. The dysfunction of lipid metabolic pathways such as sphingolipid and glycerophospholipid metabolism may be an important factor in Haematitum-induced pulmonary toxicity. This study provides a reference for further research on the mechanism of Haematitum pulmonary toxicity.

[Dielectric properties of tidal volume changes in rabbit lung tissue in the 100 MHz~1 GHz band].

This paper investigates the variation of lung tissue dielectric properties with tidal volume under in vivo conditions to provide reliable and valid a priori information for techniques such as microwave imaging. In this study, the dielectric properties of the lung tissue of 30 rabbits were measured in vivo using the open-end coaxial probe method in the frequency band of 100 MHz to 1 GHz, and 6 different sets of tidal volumes (30, 40, 50, 60, 70, 80 mL) were set up to study the trends of the dielectric properties, and the data at 2 specific frequency points (433 and 915 MHz) were analyzed statistically. It was found that the dielectric coefficient and conductivity of lung tissue tended to decrease with increasing tidal volume in the frequency range of 100 MHz to 1 GHz, and the differences in the dielectric properties of lung tissue for the 6 groups of tidal volumes at 2 specific frequency points were statistically significant. This paper showed that the dielectric properties of lung tissue tend to vary non-linearly with increasing tidal volume. Based on this, more accurate biological tissue parameters can be provided for bioelectromagnetic imaging techniques such as microwave imaging, which could provide a scientific basis and experimental data support for the improvement of diagnostic methods and equipment for lung diseases.

[Staging of COVID-19 morphological manifestations in lungs depending on disease duration]. / Stadiinost' morfologicheskikh proyavlenii COVID-19 v legkikh v zavisimosti ot davnosti zabolevaniya.

OBJECTIVE: To determine morphological alterations in lung tissues in the case of novel coronavirus infection (COVID-19) in the aspect of process staging with consideration to disease duration. MATERIAL AND METHODS: The number of COVID-19-related deaths of patients aged 55-65 years equal 17 were investigated. Serial cuts of lung tissue stained with hematoxylin and eosin, obtained from several lung fragments of each corpse, were analyzed. Morphological features previously described in literature sources were taken into account. RESULTS AND CONCLUSION: It has been established, that processes in lungs have a phasic character, furthermore they are not clearly delimited in time. In addition, there were no morphological features specific to COVID-19. Obtained results may allow forensic medical experts-histologists to predict the COVID-19 duration.

Gene expression profiles in mesenchymal stromal cells from bone marrow, adipose tissue and lung tissue of COPD patients and controls.

BACKGROUND: Chronic obstructive pulmonary disease (COPD) is characterized by irreversible lung tissue damage. Novel regenerative strategies are urgently awaited. Cultured mesenchymal stem/stromal cells (MSCs) have shown promising results in experimental models of COPD, but differences between sources may impact on their potential use in therapeutic strategies in patients. AIM: To assess the transcriptome of lung-derived MSCs (LMSCs), bone marrow-derived MSCs (BM-MSC) and adipose-derived MSCs (AD-MSCs) from COPD patients and non-COPD controls. METHODS: We studied differences in gene expression profiles between the MSC-subtypes, as well as between COPD and control using RNA sequencing (RNA-seq). RESULTS: We show that besides heterogeneity between donors, MSCs from different sources have strongly divergent gene signatures. The growth factors FGF10 and HGF were predominantly expressed in LMSCs. MSCs from all sources displayed altered expression profiles in COPD, with most pronounced significantly up- and downregulated genes in MSCs from adipose tissue. Pathway analysis revealed that the most differentially expressed genes in COPD-derived AD-MSCs are involved in extracellular matrix (ECM) binding and expression. In LMSCs, the gene that differed most strongly between COPD and control was CSGALNACT1, an ECM modulating gene. CONCLUSION: Autologous MSCs from COPD patients display abnormalities with respect to their transcriptome, which were surprisingly most profound in MSCs from extrapulmonary sources. LMSCs may be optimally equipped for lung tissue repair because of the expression of specific growth factor genes.

Telocytes protect against lung tissue fibrosis through hexokinase 2-dependent pathway by secreting hepatocyte growth factor.

Pulmonary fibrosis (PF) is one of the common manifestations of end-stage lung disease. Chronic lung failure after lung transplantation is mainly caused by bronchiolitis obliterans syndrome (BOS) and is mainly characterized by lung tissue fibrosis. Pulmonary epithelial-mesenchymal transformation (EMT) is crucial for pulmonary fibrosis. Telocytes (TCs), a new type of mesenchymal cells, play a protective role in various acute injuries. For exploring the anti-pulmonary fibrosis effect of TCs in the BOS model in vitro and the related mechanism, rat tracheal epithelial (RTE) cells were treated with transforming growth factor-ß (TGF-ß) to simulate lung tissue fibrosis in vitro. The RTE cells were then co-cultured with TCs primarily extracted from rat lung tissue. Western blot, Seahorse XF Analysers and enzyme-linked immunosorbent assay were used to detect the level of EMT and aerobic respiration of RTE cells. Furthermore, anti-hepatocyte growth factor (anti-HGF) antibody was exogenously added to the cultured cells to explore further mechanisms. Moreover, hexokinase 2 (HK2) in RTE cells was knocked down to assess whether it influences the blocking effect of the anti-HGF antibody. TGF-ß could induce lung tissue fibrosis in RTE cells in vitro. Nevertheless, TCs co-culture decreased the level of EMT, glucose metabolic indicators (lactate and ATP) and oxygen levels. Furthermore, TCs released hepatocyte growth factor (HGF). Therefore, the exogenous addition of anti-HGF antibody in the co-culture system blocked the anti-lung tissue fibrosis effect. However, HK2 knockdown attenuated the blocking effect of the anti-HGF antibody. In conclusion, TCs can protect against lung tissue fibrosis by releasing HGF, a process dependent on HK2.

New Insights via RNA Profiling of Formalin-Fixed Paraffin-Embedded Lung Tissue of Pulmonary Fibrosis Patients.

In sporadic idiopathic pulmonary fibrosis (sIPF) and pulmonary fibrosis caused by a mutation in telomere (TRG-PF) or surfactant related genes (SRG-PF), there are a number of aberrant cellular processes known that can lead to fibrogenesis. We investigated whether RNA expression of genes involved in these processes differed between sIPF, TRG-PF, and SRG-PF and whether expression levels were associated with survival. RNA expression of 28 genes was measured in lung biopsies of 26 sIPF, 17 TRG-PF, and 6 SRG-PF patients. Significant differences in RNA expression of TGFBR2 (p = 0.02) and SFTPA2 (p = 0.02) were found between sIPF, TRG-PF, and SRG-PF. Patients with low (

Fast-track preparation of lung specimens for electron microscope observations of the pulmonary endothelial glycocalyx.

The glycocalyx (GCX) covers the luminal surface of blood vessels and regulates vascular permeability. As GCX degradation predicts various types of vasculopathy, confirming the presence of this structure is useful for diagnosis. Since the GCX layer is very fragile, careful fixation is necessary to preserve its structure. We explored appropriate and feasible methodologies for visualizing the GCX layer using lung tissue specimens excised from anesthetized mice. Each specimen was degassed and immersed in Alcian blue (ALB) fixative solution, and then observed using electron microscopy. Specimens from septic mice were prepared as negative GCX controls. Using these immersion-fixed specimens, the GCX layer was successfully observed using both transmission and scanning electron microscopy; these observations were similar to those obtained using the conventional method of lanthanum perfusion fixation. Spherical aggregates of GCX were observed in the septic mouse specimens, and the GCX density was lower in the septic specimens than in the non-septic specimens. Of note, the presently reported methodology reduced the specimen preparation time from 6 to 2 days. We, therefore, concluded that our novel method could be applied to human lung specimens and could potentially contribute to the further elucidation of vasculopathies.

[Isolated bronchoplastic procedure for typical carcinoid of the left lung]. / Izolirovannaya bronkhoplasticheskaya operatsiya mezhdolevoi shpory pri kartsinoide levogo legkogo.

Lung carcinoids (LC) comprise neuroendocrine lung tumors of low (typical carcinoid) and intermediate (atypical carcinoid) grade of malignancy accounting for less than 2% of all lung neoplasms. In Europe, annual incidence of LC varies from 0.2 to 2 per 100 000. This value increased dramatically over the past 30 years. One of the causes is improvement of diagnostic methods. Compared to aggressive high-grade neuroendocrine lung cancer, natural course of early-stage LC is usually indolent. Therefore, surgery with preservation of as much normal lung tissue as possible is preferable for resectable tumors. Nevertheless, the number of isolated bronchial resections with preservation of the entire lung tissue is relatively small, and these procedures remain technically complex interventions. We present isolated resection of interlobular spur, lower medial wall of distal part of the left main bronchus and proximal part of the lower lobular bronchus for typical carcinoid with monobronchial anastomosis and preservation of the entire lung parenchyma.

Interleukin-11 receptor subunit α-1 is required for maximal airway responsiveness to methacholine after acute exposure to ozone.

Interleukin (IL)-11, a multifunctional cytokine, contributes to numerous biological processes, including adipogenesis, hematopoiesis, and inflammation. Asthma, a respiratory disease, is notably characterized by reversible airway obstruction, persistent lung inflammation, and airway hyperresponsiveness (AHR). Nasal insufflation of IL-11 causes AHR in wild-type mice while lung inflammation induced by antigen sensitization and challenge, which mimics features of atopic asthma in humans, is attenuated in mice genetically deficient in IL-11 receptor subunit α-1 (IL-11Rα1-deficient mice), a transmembrane receptor that is required conjointly with glycoprotein 130 to transduce IL-11 signaling. Nevertheless, the contribution of IL-11Rα1 to characteristics of nonatopic asthma is unknown. Thus, based on the aforementioned observations, we hypothesized that genetic deficiency of IL-11Rα1 attenuates lung inflammation and increases airway responsiveness after acute inhalation exposure to ozone (O3), a criteria pollutant and nonatopic asthma stimulus. Accordingly, 4 and/or 24 h after cessation of exposure to filtered room air or O3, we assessed lung inflammation and airway responsiveness in wild-type and IL-11Rα1-deficient mice. With the exception of bronchoalveolar lavage macrophages and adiponectin, which were significantly increased and decreased, respectively, in O3-exposed IL-11Rα1-deficient as compared with O3-exposed wild-type mice, no other genotype-related differences in lung inflammation indices that we quantified were observed in O3-exposed mice. However, airway responsiveness to acetyl-ß-methylcholine chloride (methacholine) was significantly diminished in IL-11Rα1-deficient as compared with wild-type mice after O3 exposure. In conclusion, these results demonstrate that IL-11Rα1 minimally contributes to lung inflammation but is required for maximal airway responsiveness to methacholine in a mouse model of nonatopic asthma.

Zinc metalloprotease ProA of Legionella pneumophila increases alveolar septal thickness in human lung tissue explants by collagen IV degradation.

ProA is a secreted zinc metalloprotease of Legionella pneumophila causing lung damage in animal models of Legionnaires' disease. Here we demonstrate that ProA promotes infection of human lung tissue explants (HLTEs) and dissect the contribution to cell type specific replication and extracellular virulence mechanisms. For the first time, we reveal that co-incubation of HLTEs with purified ProA causes a significant increase of the alveolar septal thickness. This destruction of connective tissue fibres was further substantiated by collagen IV degradation assays. The moderate attenuation of a proA-negative mutant in A549 epithelial cells and THP-1 macrophages suggests that effects of ProA in tissue mainly result from extracellular activity. Correspondingly, ProA contributes to dissemination and serum resistance of the pathogen, which further expands the versatile substrate spectrum of this thermolysin-like protease. The crystal structure of ProA at 1.48 Å resolution showed high congruence to pseudolysin of Pseudomonas aeruginosa, but revealed deviations in flexible loops, the substrate binding pocket S1 ' and the repertoire of cofactors, by which ProA can be distinguished from respective homologues. In sum, this work specified virulence features of ProA at different organisational levels by zooming in from histopathological effects in human lung tissue to atomic details of the protease substrate determination.